Compound Mutagenesis Screening And Active Substance Structure Identification And Its Mechanism Of Bacillus Amyloliquefaciens With Resistance To Grass Carp Aeromonas

Bacterial fish disease outbreaks has became more frequent,with the increase of aquaculture density.Aeromonas hydrophila and Aeromonas veronii have the characteristics of easy reproduction and strong pathogenicity,which caused serious economic losses to aquaculture.Therefore,the study of beneficial microorganisms is a research hotspot in the prevention and control of freshwater fish diseases and set Bacillus amyloliquefaciens as a probiotic feed additive is a common biological control method.A strain of Bacillus amyloliquefaciens X030(Ba X030)was screened from soil,whose secondary metabolites showed antibacterial activity against Aeromonas hydrophila X040(Ah X040)and Aeromonas veronii X005(Av X005).However,the yield of active substances from the strains screened in nature is very low.Therefore,it has important theoretical significance and research value to optimize the medium composition of Ba X030 and screen mutant strains with higher resistance to grass carp pathogens through physical and chemical mutagenesis.Macrolactin A,a polyketide was identified from Ba X030 fermentation product by a rapid purification system of biomolecules combined with high resolution mass spectrometry and nuclear magnetic resonance technology,its molecular weight was402 and the chemical formula was C₂₄H₃₄O₅.Another antibacterial product was identified as polyketide oxydifficidin.The molecular weight was 559,the chemical formula was C₃₁H₄₄O₇P.In order to improve antibacterial activity and optimize fermentation conditions,the yield of macrolactin A was increased from 8.12?g/m L to11.84?g/m L and 11.61?g/m L after sucrose or mannitol supplementation,and the yield of oxydifficidin was 2.29 and 2.05?g/m L,respectively.They promoted the growth and metabolic regulation of Ba X030.Oxydifficidin was not detected in the control group.The cell experiment showed that the two active substances had no toxicity to liver cells of grass carp.i TRAQ was used to identify the difference in total protein expression levels of Ba X030 cultured in different media.A total of 2055proteins were screened.KEGG analysis showed that the experimental group had the most significant enrichment in metabolic process,the key functional proteins were upregulated in pentose phosphate pathway,amino acid and fatty acid synthesis pathway,and gluconeogenesis pathway was inhibited.Glycolysis pathway and TCA cycle were enhanced in the sucrose group,and pyruvate dehydrogenase was down-regulated in the mannitol group.Proteomics comparison showed that the expression levels of genes related to macrolactin A and oxydifficidin biosynthesis(Pks2A,Pks2E,Dif A,Dif I)were upregulated,which was beneficial to the increase of their yield,the proteins related to sporulation(Spo IIIAG,Ger Q,Ger PC,Spo VM,Spo IIAA)were down-regulated to varying degrees,and the sporulation was delayed.We firstly discovered the influence of some potential regulatory proteins(Sec G,Prk A,Mec G,Com G)on macrolactin A.The transcription levels of nine key differential genes(leu A,leu D,zwf,fab D,sec G,pdk2,pck A,ger D,ger PC)were analyzed by q RT-PCR,the results showed that the transcription levels were positively correlated with the protein expression levels.To further improve the effect of active substances against grass carp disease,compound mutagenesis of atmospheric and room temperature plasma(ARTP)and nitrosoguanidine(NTG)was carried out in Ba X030.After two round of ARTP(optimal time:30 s-60 s)mutagenesis compound with one round of NTG(optimal concentration:200-300?g/m L)mutagenesis,a mutant strain Ba N-11 with high yields of macrolactin A and oxydifficidin was obtained,which were 2.01 and 3.68 times higher than Ba X030,respectively.Scanning electron microscope observed that the morphology of Ba N-11 became elongated.Multiple SNP mutations and In Del mutations were found in gene clusters associated with active substance synthesis by resequencing.Seven regulatory factors(fab D,dif G,pks2F,pts A,nag R,fba A,pdk2)were selected for q RT-PCR,and they were up-regulated in different degrees.Single factor experiments showed that sucrose,beef extract and glutamic acid could promote the biosynthesis of oxydifficidin.Response surface experiments demonstrated that the yield of oxydifficidin in Ba N-11 significantly increased to 13.54?g/m L when cultured in the optimized culture medium.The effects of Ba N-11 on the intestinal flora of grass carp were analyzed by the third-generation high-throughput sequencing technology.After feeding Ba N-11,the abundance of Proteobacteria and Fusobacteria in intestinal flora showed a comparative advantage,the relative abundance of Rhodobacteraceae,Nocardioidaceae and Geobacteraceae were increased,and the ratio of Planctomycetes and Actinomycetes were significantly decreased,which improved the intestinal microbial constitute of grass carp.The vitro and vivo experimental results showed that Ba X030 and Ba N-11 supplementation can reduce pathological changes in liver,kidney and body surface of grass carp,and also obviously increase the expression of immune factors(Ig M,C3,LSZ,IL-1?,IL8)in liver and kidney.Compared to Ba X030,Ba N-11 showed a significantly reduce of grass carp mortality.This is the first report that Ba X030 has been subjected to ARTP-NTG compound mutagenesis to obtain a mutant strain N-11 with high yield of macrolactin A and oxydifficidin,which has higher resistance to Ah X040 and Av X005 than Ba X030.The mutation sites of the gene cluster and the change in metabolic regulation pathways were found by resequencing.The key functional proteins and potential regulatory factors in the biosynthesis process were discovered by predicting the biosynthesis gene cluster of isolated and identified antibacterial active compounds.As probiotics,Ba X030 and Ba N-11 enhanced the immunity of grass carp and affected the intestinal microflora to better adapt to the changing environment.The results revealed their potential application value in aquaculture,which can be used to control bacterial diseases,and lay an important theoretical basis and technical approach for healthy breeding in grass carp.